UHPLC‐ESI‐QTOF‐MS2 analysis of Acacia pennata extract and its effects on glycemic indices, lipid profile, pancreatic and hepatorenal alterations in nicotinamide/streptozotocin‐induced diabetic rats

Abstract Diabetes mellitus (DM) is a chronic disorder associated with severe metabolic derangement and comorbidities. The constant increase in the global population of diabetic patients coupled with some prevailing side effects associated with synthetic antidiabetic drugs has necessitated the urgent need for the search for alternative antidiabetic regimens. This study investigated the antidiabetic, antioxidant, and pancreatic protective effects of the Acacia pennata extract (APE) against nicotinamide/streptozotocin induced DM in rats. The antidiabetic activity of APE was evaluated and investigated at doses of 100 and 400 mg/kg body weight, while metformin (150 mg/kg bw) was used as a standard drug. APE markedly decreased blood glucose level, homeostatic model assessment for insulin resistance, serum total cholesterol, triglycerides, low‐density lipoprotein, blood urea nitrogen, creatinine, alanine transaminase, aspartate transaminase, and alanine phosphatase levels. Additionally, treatment with APE increased the body weight, serum insulin concentration, and high‐density lipoprotein. Moreover, activities of pancreatic superoxide dismutase, catalase, and glutathione peroxidase were increased, while the altered pancreatic architecture in the histopathological examination was notably restored in the treated rats. Ultra‐high performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight mass spectrometry (UHPLC‐ESI‐QTOF‐MS) analysis of APE showcases the prevailing presence of polyphenolic compounds. Conclusively, this study showed the beneficial effects of the Acacia pennata in controlling metabolic derangement, pancreatic and hepatorenal dysfunction in diabetic rats.


| INTRODUC TI ON
Diabetes mellitus (DM) is one of the most common metabolic diseases that cut across all age groups and ethnicity. DM is clinically defined as hyperglycemia and it is accompanied by severe deteriorating comorbidities including diabetic nephropathy, neuropathy, reproductive dysfunction, foot ulcer, retinopathy, cardiovascular diseases, depression, and Alzheimer's disease Song et al., 2021). Statistically, 463 million adults were diabetic in 2019 and the global population of diabetes will increase to 700 million by 2045, and 90% of these reported cases is attributed to type 2 diabetes (IDF Report, 2019). In addition, several millions of people living with diabetes are undiagnosed or are not even aware of their diabetic status (International Diabetes Federation, 2019). Aside the public health burden that DM poses, the significant economic burden associated with DM is very huge. Over 760 billion dollars were expended on diabetes, and it is estimated that 845 billion dollars would be utilized globally for diabetes health-related expenditure (Williams et al., 2020). A relatively sizeable population of people living with diabetes are low-income earners or live in developing nations, where the huge financial burden associated with the treatment of DM is almost not affordable. Furthermore, the antidiabetic drugs currently in clinical use are either not affordable or accessible to most diabetic patients, in addition to the various side effects associated with these drugs (Olatunji et al., 2018;Song et al., 2021).
These limitations further increase the morbidity and mortality rates associated with DM. As such, the search for affordable, easily accessible, safer, and indigenous antidiabetic regimens is imperative.
In many countries across the world especially in Asia and Africa, the use of medicinal plants or herbs constitutes a vital part in primary health care due to either religious or cultural beliefs.
Specifically, numerous studies have highlighted the relevance of medicinal plants in the treatment of DM and DM-associated comorbidities (Andrade et al., 2020;Farzaei et al., 2017). Additionally, the perceived safety and efficacy index of medical plants has increased their relevance across the globe. Acacia pennata (family: Mimosaceae) is a perennial herb vegetable domiciled in several countries including China, Thailand, Myanmar, and Bangladesh (Kim et al., 2015). In Thailand, the plant is widely used in Thai cuisine. A. pennata is rich in terpenoids and flavonoid glycosides.
Traditionally, the plant is used alone or in combination with other plants for treating diabetes, cough, fever, headaches, snake bites, scorpion stings, and rheumatism (Andrade et al., 2020). Regarding pharmacological studies, A. pennata has been reported to possess significant antinociceptive, antioxidant, antimicrobial, antiinflammatory, α-glucosidase and α-amylase inhibition as well as anti-Alzheimer's properties (Andrade et al., 2020;Dongmo et al., 2005Dongmo et al., , 2007Lomarat et al., 2015). However, studies relating to the antidiabetic effects of the plant are still lacking and there are no scientific reports available to validate its traditional use as an antidiabetic regimen. Therefore, this study investigated the antidiabetic, antihyperlipidemic, and antioxidant effects of A. pennata extract in diabetic rats.

| Plant material
The aerial part of A. pennata was purchased from a local vegetable market in Hat Yai (Songkla, Thailand) in June 2020, and the identification was performed at the Faculty of Thai Traditional Medicine, Prince of Songkla University, Thailand. A reference sample (#FBRSVP0000270438) was kept at the herbarium of the faculty.

| Drugs and chemicals
Metformin hydrochloride, nicotinamide, and streptozotocin were purchased from Alfa Aesar. All other chemicals and reagents used were of analytical grade.

| Preparation of Acacia pennata extract (APE)
The plant was thoroughly cleaned under running tap water and oven dried for 4 days. The dried aerial part of the plant was pulverized with a mechanical grinder. Five hundred grams of the powdered sample was macerated in 70% ethanol at a solvent/solute ratio of 10:1 (v/w) for 24 h on a shaker. The extracted solution was filtered and concentrated to 30% of the original solution using a rotary evaporator. The resulting solution was dechlorophyllized using the sedimentation procedure Olatunde, Tan et al., 2021;). Briefly, the solution of the extract was kept overnight at 4°C. Thereafter, the sample was centrifuged at 10,000 g for 30 min at 4°C. The supernatant was collected and lyophilized. The light brown extract was termed "APE" and stored until further use.

| UHPLC-DAD-ESI-QTOF-MS profiling of Acacia pennata extract
UPLC-ESI-Q-TOF-MS analysis of APE was performed by solubilizing 50 mg of APE powder in 1 ml of 50% aqueous methanol. The resulting solution was centrifuged and the clear supernatant obtained was subjected to UHPLC-ESI-QTOF-MS analysis using the previously reported protocol .

| Animals, establishment of diabetes, and treatment
Specific pathogen-free male rats (7 weeks old; 185 ± 20 g) were used for the in vivo antidiabetic experiment. The animals were accommodated in stainless cages and fed with standard rat chow and water ad libitum. Additionally, the animals were maintained at a temperature of 22 ± 2°C, relative humidity of 55 ± 5%, and a 12 h light and dark cycle to simulate natural rhythm. The experimental protocol used in this study conformed to the guidelines of the National Institutes of Health (NIH, revised 1979) and was approved by the Ethics

Committee of the East China Normal University Affiliated Wuhu
Hospital (approval number, Wuhuey/2021/0926). The animals were acclimatized to the experimental environment for 1 week.
Thereafter, type 2 diabetes mellitus was induced in overnight fasted rats by administering an intraperitoneal injection of 150 mg/kg of nicotinamide 30 min before administering streptozotocin (65 mg/ kg, i.p.; Abdel Aziz et al., 2020;Ojuade et al., 2021). Seventy-two hours poststreptozotocin administration, the fasting blood glucose (FBG) level of the overnight fasted rats was determined using Accu- Chek guide glucometer to confirm the establishment of DM. Rats with FBG concentration above 250 mg/dl were adjudged diabetic and incorporated into the study. Diabetic rats were then randomly assigned into four groups of six rats each as follows: Diabetic control rats (DMR): treated with normal saline.
Six healthy nondiabetic rats were designated as normal control (HNR) and were administered normal saline. The doses of the extract and metformin used were adopted from previous studies (Dongmo et al., 2005;Xiang et al., 2021). All the groups were administered their respective treatments once a day per os for 4 weeks. A weekly measurement of the body weight and FBG concentration was performed, while the water and food intake was determined on a daily basis.

| Posttreatment intraperitoneal glucose tolerance test (IPGTT)
After the treatment, the rats were fasted overnight and a solution of 2 g/kg glucose was intraperitoneally injected into each rat from all the experimental groups. The blood glucose concentration was determined from the blood sample taken from the tail vein of each rat at an interval of 30 min for 120 min .

| Animal sacrifice and biochemical analysis
The rats were anesthetized under thiopental sodium and blood was collected via cardiac puncture, centrifuged to obtain the serum that was used for the determination of lipid profiles, hepatorenal biomarkers, and insulin levels. The pancreas was excised after animal sacrifice for histopathology using routine hematoxylin and eosin (H&E) procedures.

| Determination of serum biochemical parameters
The serum obtained from the blood samples after centrifugation was used for the determination of serum lipids (triglycerides (TG), total cholesterol (TC), LDL-C, and HDL) and hepatorenal biomarkers (ALT, AST, ALP, creatinine, BUN, and uric acid) using an automated chemistry analyzer ).

| Determination of hemoglobin A1c and fasting plasma insulin
Hemoglobin A1c (HbA1c) and fasting plasma insulin (FINS) were determined with enzyme-linked immunosorbent assay (ELISA) kits from the Nanjing Jiancheng Bioengineering Institute (China) according to the protocols of the manufacturer, while HOMA-IR was determined using the formula stated below:

| Pancreas histological assessment
The pancreas was preserved in 10% neutral buffered formalin solution was used for the histological assessment using routine standard hematoxylin and eosin (H&E) procedures, as previously described .

| Statistical analysis
GraphPad Prism 5.0. was used for statistical analysis, and data were presented as mean ± SD (n = 6). Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by a Dunnett post hoc test for comparison between experimental groups. The value of p < .05 was considered significant.

| Identification of secondary metabolites in Acacia pennata extract
The identification of the bioactive metabolites in APE was carried out using UHPLC-ESI-QTOF-MS/MS ( Figure 1). As shown in Table 1, APE has a robust polyphenolic content ranging from simple phenolic compounds like trolox, to flavones such as baicalein and luteolin.

| Effect of APE on blood glucose concentration in DM rats
The average FBG concentration in the HNR (95.0 mg/dl) was significantly lower than the DMR rats (420.3 mg/dl) at day 0 of the treatment. At the end of the treatment period (day 28), the FBG level of the HNR was 99.3 mg/dl, which was significantly lower than the corresponding FBG level of the DMR at the same period (509.5 mg/ dl). During the course of the treatment, the blood glucose level of the DMR group was consistently and significantly higher than the normal rats ( Figure 1a). Whereas in diabetic rats treated with APE (100 and 400 mg/kg), there was a dose-dependent and significant reduction of the FBG concentration to 266.1 and 198.5 mg/dl, respectively, compared to the DMR group at the end of the treatment ( Figure 2a).

| Effect of APE on body weight loss, food and water intake in DM rats
Diabetes mellitus significantly increased the food and water consumption of the DMR group when compared to the HNR group (Figure 2b-c). Conversely, treatment of diabetic rats with APE (100 and 400 mg/kg) resulted in significant reduction in the food and water intake of the treated groups compared to the DMR (Figure 1b).
Despite the significant increase in the food intake of the DMR group, there was a 17% reduction in the body weight at the end of the study, which was markedly lower than that of the HNR group. The HNR has a 62.6% increase in their body weight ( Figure 1c). However, treatment with APE (100 and 400 mg/kg) significantly increased the bodyweight gain of the treated rats by 29.3 and 33.7%, respectively ( Figure 2d).

| Effect on glucose and insulin sensitivity in DM rats
In the IPGTT, the blood glucose concentration of the DMR group was consistently and markedly higher at all the measured times (0, 30, 60, 90, and 120 min),compared to the corresponding values of the rats in the HNR group. Whereas, the glucose sensitivity was significantly reduced in the diabetic rats treated with APE from 60 to 120-min postglucose administration compared to the DMR group ( Figure 3a). Additionally, the DMR rats showed significantly reduced FINS levels compared to the HNR group, but significantly improved in APE-treated groups ( Figure 3b). Furthermore, HOMA-IR and HbA1c were observed to be significantly increased in the diabetic control group, while treatment of diabetic rats with APE markedly decreased HOMA-IR and HbA1c compared to the DMR group (Figure 3c-d).

| Effect on the serum biochemical parameters in DM rats
The results presented in Figure 4 indicate that the serum lipids including total cholesterol, triglycerides. and low-density lipoprotein cholesterol (LDL-C) levels in the DMR group were drastically increased (161, 206, and 68.1 mg/dl, respectively), while HDL was reduced compared to the corresponding values in the HNR group (71.3, 69, and 18.8 mg/dl, respectively). Additionally, the serum hepatorenal biomarkers, including ALT, AST, ALP, BUN, creatinine, and uric acid, were significantly increased relative to the HNR group (Figure 5a-f). Conversely, treatment of diabetic rats with APE significantly ameliorated these serum biochemical alterations when compared to the DMR group (Figures 4 and 5).

| Effect on pancreas histology in DM rats
The representative H&E-stained pancreas of the HNR group indicated significant number and size of the islet of Langerhans, compared to the DMR group with a reduced islet size as well as necrotic acinar cells (Figure 6a,b). The representative images from the pancreas of APE (100 and 400 mg/kg) and metformin-treated groups showed restored islet of Langerhans cellularity, as indicated by increased size of the islets as well as improved pancreas acinar cells ( Figure 6).

| Effect on oxidative stress biomarkers in DM rats
As shown in Table 2, DM markedly increased pancreatic MDA level in the DMR group compared with the HNR. However, the administration of APE and metformin significantly lowered MDA levels in the pancreas compared with the DMR group. The modulatory effects of APE treatment on antioxidant capacity in diabetic rats are also portrayed in Table 2. In line with the MDA results, the activities of antioxidant enzyme biomarkers including SOD, GPx, and CAT were significantly declined in the pancreatic tissues of the DMR group.
In contrast, the rats treated with APE and metformin displayed significant increases in GPx, SOD, and CAT activities in the pancreatic tissues of the treated rats compared to the DMR group (Table 2).  that diabetic rats presented impaired kidney, liver, and pancreatic function as well as increased oxidative stress in vital organs (Gao et al., 2021;Makinde et al., 2020;Olatunji et al., 2017). The results of the present study showed that treatment with APE significantly F I G U R E 3 Effect of the Acacia pennata extract (APE) on intraperitoneal glucose tolerance test (IPGTT), fasting plasma insulin (FINS), homeostatic model assessment for insulin resistance (HOMA-IR), and hemoglobin A1c (HbA1c) levels in nicotinamide/streptozotocin (NICO/STZ)-induced diabetic rats. Values were expressed as mean ± SD (n = 6). **p < .001 compared with the healthy nondiabetic rats (HNR) group. ##p < .001, &&p < .01, $$p < .05 compared with the diabetic control rats (DMR) group Under normal physiological state, the body converts excessive glucose in the blood to glycogen through the stimulation of insulin.

| DISCUSS ION
Whereas, in type 2 diabetes, insulin secretion/sensitivity is impaired F I G U R E 5 Effect of the Acacia pennata extract (APE) on serum alanine transaminase (ALT), aspartate transaminase (AST), alanine phosphatase (ALP), blood urea nitrogen (BUN), creatinine, and uric acid in nicotinamide/streptozotocin (NICO/STZ)-induced diabetic rats. Values were expressed as mean ± SD (n = 6). **p < .001 compared with the healthy nondiabetic rats (HNR) group. ##p <.001, &&p <.01, $$p <.05 compared with the diabetic control rats (DMR) group high incidence of cardiovascular diseases in diabetic patients (Mollica et al., 2017;Tan et al., 2006). Thus, regulating insulin secretion can improve glucose conversion, synthesis of lipids, and blood glucose level. The result obtained from this study indicated that APE markedly increased insulin secretion, islets cellularity, and ameliorated pancreatic architecture in diabetic rats, which in turn led to decrease in the blood glucose and lipid profile concentrations. Treatment with APE resulted in significant reduction in blood glucose, TG, TC, and LDL-C levels.
The oral administration of APE significantly altered diabetesinduced hepatorenal dysfunction, as indicated by a reduction in functional biomarkers of the liver (ALT, AST, and ALP) and kidney (BUN, uric acid, and Scr). Diabetes has been strongly implicated in liver and kidney abnormalities. The release of markers of hepatotoxicity including ALP, AST, and ALT into the blood suggests hepatic injury (Adefegha et al., 2014). The liver is critically involved in several physiological processes including insulin clearance, glucose regulation, lipid and carbohydrate metabolism, and deficiencies in insulin function can cause liver abnormalities due to gluconeogenesis, resulting in increased serum liver enzymes (Ghosh & Suryawanshi, 2001). Furthermore, diabetic rats showed increased levels of typical biomarkers of renal injury including high levels of serum BUN, urea, and creatinine which are consistent with previous studies (Kishore et al., 2017;Kpemissi et al., 2020;Zhang et al., 2020).  Values were expressed as mean ± SD (n = 6). Data were analyzed by the analysis of variance (ANOVA) followed by Dunnett's test.
*p < .001 compared with the HNR group. **p < .001 compared with the DMR group.
Numerous studies have indicated that diabetes-induced oxidative damages can effectively be mitigated by polyphenolic constituents due to their ability to interfere in the pathophysiology of the disease (Cao et al., 2019;Domínguez Avila et al., 2017;Solayman et al., 2016;Sun et al., 2020). As such, the synergistic hypoglycemic and antioxidant properties of these metabolites may possibility account for the ameliorative effects of APE on the diabetic rats.

| CON CLUS ION
Taken together, the results presented in this study portrayed the therapeutic effect of APE against NICO/STZ-induced DM. This is evidenced by the ability of APE to alleviate glucose and insulin homeostasis, ameliorate metabolic derangement including dyslipidemia, hepatorenal dysfunction, and restore pancreatic oxidative stress and β-cell function in the treated diabetic animals. These findings may enhance a better understanding of the therapeutic effect of A. pennata on diabetes and the possible development of the plant as for future antidiabetic application. However, further molecular studies are needed to further shed deeper insight into the antidiabetic mechanism of APE.

ACK N OWLED G M ENT
The authors are grateful to East China Normal University Affiliated Wuhu Hospital.

CO N FLI C T O F I NTE R E S T
None declared by the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.